FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease.
FRET-based kinetic analysis of highly reactive heterochiral DNA toward EcoRI endonuclease.
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DOI：
10.1016/j.bbrc.2009.08.164
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发表时间：
2009-12
影响因子：
3.1
通讯作者：
H. Urata;Chihiro Tamaki;M. Matsuno;S. Wada;M. Akagi
中科院分区：
文献类型：
--
作者：
H. Urata;Chihiro Tamaki;M. Matsuno;S. Wada;M. Akagi
我们发现了一个异手性十二烷基氧基核苷酸对EcoRI的异常反应性，该核苷酸在六聚体EcoRI识别序列的3 '侧位含有一个非天然的l-核苷酸残基。在本研究中，通过一个异手性分子信标确定了反应的动力学参数，该分子信标在茎区含有EcoRI识别序列，在5 ‘端和3 ’端分别具有荧光和猝灭染料。我们发现，与相应的同手性分子信标相比，异手性分子信标具有较大的vmax和较小的km值。这种手性修饰有望诱导更适合EcoRI结合和催化的构象变化。
We found unusual reactivity of a heterochiral dodecadeoxynucleotide toward EcoRI that contains an unnatural l-nucleotides residue at the 3′-flanking site of the hexameric EcoRI recognition sequence. In this study, the kinetic parameters for the reaction were determined by means of a heterochiral molecular beacon that contains the EcoRI recognition sequence in the stem region and possesses the fluorescent and quenching dyes at the 5′- and 3′-termini, respectively. We found that the heterochiral molecular beacon showed large Vmaxand small Kmvalues compared to the corresponding homochiral one. This chiral modification is expected to induce a preferable conformational change for binding and catalysis by EcoRI.